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We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml-1 Con A, 10 U ml-1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II3Neu-Gg4Cer, Gg4Cer and IV3GalNAc alpha-Gb4Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II3Neu-Gg3Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV3Neu-Gg5Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc beta 1-4(NeuAc or NeuGc alpha 2-3)Gal beta-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation of N-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

We have cloned a cDNA encoding beta 1-4N-acetylgalactosaminyltransferase (EC 2.4.1.92) (GalNAc-T) from rat ascites hepatoma of the free-cell type AH7974F. The cell line only expressed asialo-series glycosphingolipids (GSLs) including asialo-GM2 [Taki, T., Hirabayashi, Y., Ishiwata, Y., Matsumoto, M., and Kojima, K. (1979) Biochim. Biophys. Acta 572, 113-120]. The cDNA, pGNA56, was isolated by screening AH7974F cDNA library in lambda gt10 with a probe. The probe was obtained from AH7974F cDNA by PCR using primers with the nucleotide sequence of the human GalNAc-T cDNA. The amino acid sequence deduced from the nucleotide sequence of pGNA56 exhibited 88% similarity to the human GalNAc-T sequence. The enzyme was a typical type II membrane protein, which consisted of a short N-terminal residue, a transmembrane region, and a long C-terminal residue, including the catalytic domain. The substrate specificity of rat GalNAc-T was determined using homogenates from cells into which the cDNA clone was transfected. The enzyme catalysed not only the formation of GM2 and GD2 from GM3 and GD3 respectively, but also asialo-GM2 from CDH. It also acted on GSL substrates, including GM1b, sialylparagloboside and GD1 alpha. On the other hand, the enzyme did not transfer GalNAc to soluble substrates such as glycoproteins and oligosaccharide. The GSL compositional and immunocytochemical analyses of stable transfectants obtained by transfection of the cDNA showed simultaneous expression of asialo-GM2 and GM2 on the plasma membrane. Therefore, we concluded that the formation of asialo-GM2, GM2 and GD2 was catalysed by the single GalNAc-T. Northern-blot hybridization showed that the GalNAc-T mRNA was strongly expressed in rat brain, testis, and spleen. The gene was also expressed in rat normal liver to a lesser extent. We found the GSLs in asialo- and alpha-pathways such as asialo-GM1 and GD1 alpha in the rat tissues by using a sensitive t.l.c.-immunostaining method. These observations also supported our conclusion that the single GalNAc-T synthesizes asialo-GM2, GM2 and GD2 in vivo.

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